The long-term objective of this project is to elucidate mechanisms of ion transport across the inner membrane of mitochondria. Specific aims are: (1) To study the mechanism and regulation of the uncoupling protein (UcP) of brown adipose tissue mitochondria both in intact mitochondria and in proteoliposomes reconstituted with the purified 32 kDa uncoupling protein. Brown adipose tissue provides heat to small mammals and human infants, who are very susceptible to cold stress. The mechanism of heat production is broadly understood and relies on the regulated transport of H+ ions through UcP, an energy-dissipating (therefore heat-producing) protein found uniquely in this tissue. This project introduced fluorescent probe spectroscopy to study transport by UcP, a powerful technique that has produced major new findings and should lead to rapid advances in our understanding of this unique protein. (2) To study the function of mutagenized UcP. UcP belongs to an important gene family that includes the phosphate transporter and adenine nucleotide exchanger of mitochondria. Although its amino acid sequence is known, it has not been possible to exploit this information because so little is known about its transport properties. Armed with new insights into its function and transport kinetics, this project will begin a study of the chemistry of ligand interaction on a molecular level. This study will use mutagenized UcP supplied by a collaborator who has successfully overexpressed the functionally active protein in yeast cells using UcP cDNA in a yeast expression vector. (3) To study the pleiotropic effects of local anesthetics on mitochondrial ion transport and to resolve their mechanism of uncoupling. Bupivicaine is a new local anesthetic that can produce serious toxicity in patients. It has been found to uncouple oxidative phosphorylation in mitochondria, and this has been a suggested basis of its toxicity. There is controversy in the literature over the mechanism of uncoupling, and this project will attempt to resolve this by measurements of ion transport and respiration of mitochondria.